Many chemotherapy drugs act against cancer cells by causing damage to the DNA. Resistance to chemotherapy is a major clinical obstacle in cancer treatment. The mechanisms of chemoresistance in cancer patients are not fully understood, leading to urgent needs for determining factors that control drug response and developing novel therapies to enhance the treatment efficacy. Signaling from transforming growth factor (TGF) , a tumor suppressor in normal cells, is hijacked in cancer to promote disease progression. In breast cancer, TGF- is linked to poor clinical outcomes and chemoresistance through mechanisms that remain largely unknown. Our previous studies indicate that in breast cancer cells, TGF- induces microRNAs (miR-21 and miR-181) that target the DNA damage sensors ATM and MSH2, and may therefore regulate cancer response to genotoxic chemotherapy. The goals of this study are to dissect the molecular mechanism of TGF-2-mediated chemoresistance, and to explore potential therapies to enhance drug efficacy. In Aim 1, TGF- action on cell response to various DNA-damaging treatments and to inhibition of poly(ADP-ribose) polymerase (PARP) will be determined in breast cancer cells with different p53 status using established molecular and cellular biology assays. The role of the TGF- -regulated miRNAs and the ATM/MSH2 pathways will be determined using gene knockdown and overexpression strategies. In Aim 2, the hypothesis that enhanced SMAD2/3 binding to their RNA targets mediates TGF- 's functional shift in cancer cells towards inducing miRNA regulation and chemoresistance will be examined. Breast cancer cells expressing various levels of the SMAD2/3 cofactors (i.e., SMAD4, Drosha and p68) will be examined for their dynamic regulation of SMAD2/3 function and TGF-2 effect. In Aim 3, the effect of TGF- on chemotherapy response and the mechanism identified in the first two aims will be evaluated in animal tumor models. Novel strategies to therapeutically suppress this TGF- function and enhance the treatment efficacy will be explored. This study will enable better understandings of drug resistance and of TGF- signaling as both a marker and a target in cancer treatment. Although the mechanism identified herein may have a general application to understanding cancer and defining treatments, our study has added significance for clinically aggressive, hard-to-treat basal- like (mainly triple-negative) breast cancer that often experience active TGF- signaling. This study will provide novel insight into the functional switch of TGF- in cancer via SMAD2/3-mediated miRNA processing. Understanding TGF-2-mediated chemoresistance may reveal novel therapeutic targets and strategies that will enhance the chemotherapy efficacy for cancers that lack targets for systemic treatments. Our long-term objectives are to validate this mechanism in primary cancers and establish standard approaches to identify patients suitable for therapies targeting TGF- 's drug resistant effect, and to understand the global effect of TGF- -mediated miRNA dysregulation in human cancer.